Understanding mechanisms underlying control of tumorigenesis and metastasis is central to development of means to intervene in these processes. Reports from three laboratories suggest that the Siah ubiquitin ligases (Siahl and Siah2) function in pancreatic, mammary, melanoma and prostate tumor development and/or progression. Our studies revealed the role of Siah2 in melanoma development and progression through its effect on HIF and Ras signaling pathways. By regulating prolyl hydroxylase 3 stability, Siah2 controls HIFIa availability and the ability of melanoma cells to metastasize, without affecting tumorigenicity. By regulating Sprouty2 (SPRY2) stability, Siah2 regulates Ras and Raf signaling pathways, which dictate melanoma tumorigenicity. Consistent with these findings, Siah2 expression increases in more mestastatic melanomas, as determined by analysis of melanoma TMA. These findings provide a rationale to investigate mechanisms underlying regulation and function of Siah2 in melanoma. Our initial observations also suggest that AKT regulates Siah2 transcription. We will delineate mechanisms underlying Akt-mediated increase of Siah2 expression in melanoma where 50% of tumors contain a constitutively active Akt. Collectively, our findings indicate that: (i) Siah2 expression is upregulated in malignant melanomas, (ii) Siah2 inhibition attenuates melanoma tumorigenicity through SPRY2-Ras signaling, (iii) Siah2 inhibition attenuates melanoma metastasis through PHD3-HIF signaling, and (iv) AKT regulates Siah2 expression. Together, these observations provide the foundation for our hypothesis that under the control of AKT signaling, Siahl/2 plays a central role In regulation of melanoma tumorigenesis and metastasis. To test this hypothesis we will use biochemistry, molecular biology, cell biology and mouse models to: (1) extend our original findings defining AKT-dependent mechanisms underlying regulation of Siah2 transcription, and (2) characterize Siah2-Sprouty2 interaction relevant to different stages of melanoma tumor development. We will also (3) utilize Tg-N-Ras/Alnk4a mice, which develop metastatic melanoma, to assess the role of Siah (following crosses with Siah1a'':Siah2''and Siah2':Siah1a*'' mice) in melanoma development and progression. Additional studies addressing Siah2 activity in tumorigenesis and metastasis will be carried out in collaboration with Project 2 to identify metabolic pathways affected by Siah2 and with Project 3, which focuses on identification and characterization of Siah2 inhibitors. Collectively this project should provide new important information regarding the role of Siah2 in regulating melanoma tumorigenesis and metastasis.